1. Field of the Invention
The invention is in the field of organic and medicinal chemistry. In particular, the invention relates to pyrimidines and uses thereof, such as inhibiting the activity of lysophosphatidic acid acyltransferase β (LPAAT-β) activity and/or inhibiting the proliferation of a cell (e.g., tumor cell).
2. Description of the Related Art
Lysophosphatidic acid acyltransferase (LPAAT) catalyzes the acylation of lysophosphatidic acid (LPA) to phosphatidic acid. LPA is the simplest glycerophospho-lipid, consisting of a glycerol molecule, a phosphate group, and a fatty acyl chain. LPAAT adds a second fatty acyl chain to LPA, producing phosphatidic acid (PA). PA is the precursor molecule for certain phosphoglycerides, such as phosphatidylinositol, and diacylglycerols, which are necessary for the production of other phosphoglycerides, such as phosphatidylcholine, and for triacylglycerols, which are essential biological fuel molecules.
In addition to being a crucial precursor molecule in biosynthetic reactions, LPA has been added to the list of intercellular lipid messenger molecules. LPA interacts with G protein-coupled receptors, coupling to various independent effector pathways including inhibition of adenylate cyclase, stimulation of phospholipase C, activation of MAP kinases, and activation of the small GTP-binding proteins Ras and Rho (Moolenaar, J. Biol. Chem. 28:1294 (1995)). The physiological effects of LPA have not been fully characterized as yet. However, one of the physiological effects that is known is that LPA promotes the growth and invasion of tumor cells. It has been shown that the addition of LPA to ovarian or breast cancer cell lines induces cell proliferation, increases intracellular calcium levels, and activates MAP kinase (Xu et al., Biochem. J. 309:933 (1995)). In addition, LPA has been shown to induce MM1 tumor cells to invade cultured mesothelial cell monolayers (Imamura et al., Biochem. Biophys. Res. Comm. 193:497 (1993)).
Like LPA, PA is also a messenger molecule. PA is a key messenger in a common signaling pathway activated by proinflammatory mediators such as interleukin-1β, tumor necrosis factor ax, platelet activating factor, and lipid A (Bursten et al., Am. J. Physiol. 262:C328 (1992); Bursten et al., J. Biol. Chem. 255:20732 (1991); Kester, J. Cell Physiol. 156:317 (1993)). PA has been implicated in mitogenesis of several cell lines (English, Cell Signal 8:341 (1996)). PA level has been found to be increased in either ras or fps transformed cell lines compared to the parental Rat2 fibroblast cell line (Martin et al., Oncogene 14:1571 (1997)). Activation of Raf-1, an essential component of the MAPK signaling cascade, by extracellular signals is initiated by association with intracellular membranes. Recruitment of Raf 1 to membranes has been reported to be mediated by direct association with phosphatidic acid (Rizzo et al., J. Biol. Chem. 275:23911-8 (2000)). Thus, LPAAT, as an enzyme that regulates PA content in cells, may play a role in cancer, and may also mediate inflammatory responses to various proinflammatory agents.
LPAAT exists in a LPAAT-α form and a LPAAT-β form. Northern blot analysis shows that LPAAT-(α is expressed in all human tissues tested with the highest expression level found in skeletal muscle (West et al., DNA Cell Biol. 16:691 (1997)). The uniformity of LPAAT-(α expression has also been found in additional tissues such as prostate, testis, ovary, small intestine, and colon (Stamps et al. Biochem. J. 326:455 (1997)) as well as in mouse tissues (Kume et al., Biochem. Biophys. Res. Commun. 237:663 (1997)). A 2 kb and a 1.3 kb forms, possibly due to alternative utilization of polyadenylation signals at the 3′-UTR, have been found in murine LPAAT-α mRNA (Kume et al., Biochem. Biophys. Res. Commun 237:663 (1997)), whereas only one major human LPAAT-α mRNA of 2 kb in size has been detected by Northern analysis (West et al., DNA Cell Biol. 16:691 (1997); Stamps et al., Biochem. J. 326:455 (1997)).
In contrast, LPAAT-β demonstrates a distinct tissue distribution of mRNA expression (West et al., DNA Cell Biol. 16:691 (1997)). LPAAT-β is most highly expressed in liver and heart tissues. LPAAT-β is also expressed at moderate levels in pancreas, lung, skeletal muscle, kidney, spleen, and bone marrow; and at low levels in thymus, brain and placenta. This differential pattern of LPAAT-β expression has been confirmed independently (Eberhardt et al., J. Biol. Chem. 272:20299 (1997)) with the only discrepancy being that high level, instead of moderate level, of LPAAT-β has been detected in pancreas, possibly due to slight lot variations in commercial RNA blots (Clontech, Palo Alto, Calif.). In addition, moderate LPAAT-β expression has been found in prostate, testis, ovary, small intestine, and colon with the small intestine containing relatively higher amounts (Eberhardt et al, J. Biol. Chem. 272:20299 (1997)). Within various brain sections, high expression has been found in the subthalamic nucleus and spinal cord; and least in the cerebellum, caudate nucleus, corpus callosum, and hippocampus. LPAAT-β can also be detected in myeloid cell lines THP-1, HL-60, and U937 with the mRNA levels remaining the same with or without phorbal-ester treatment. The size difference between human LPAAT-α and LPAAT-β mRNA is consistent with the sequence data, in which LPAAT-(α has a longer 3′-UTR. The differential tissue expression pattern of LPAAT-α and LPAAT-β mRNA would suggest these two genes are regulated differently and are likely to have independent functions. Therefore, a desirable feature in compounds that inhibit LPAAT activity is that they are specific in inhibiting one isoform of the enzyme over the other (i.e., LPAAT-β over LPAAT-α).
LPAAT-β mRNA has been found to be elevated in tumor tissues (e.g., uterus, fallopian tube, and ovary), as compared to its expression in the corresponding normal tissues. However, no significant difference was found in LPAAT-α mRNA level between the various tumor tissues and the normal adjacent tissues. In two of the tumor tissues (fallopian tube and ovary) where LPAAT-α mRNA was elevated, PAP2-α mRNA expression was found to be suppressed, as it was also in tumors of the colon, rectum, and breast. Thus, LPAAT-β (rather than LPAAT-α) appears to be a relevant target for inhibition.
There is a need in the art for improved compositions and methods. The present invention fills this need, and further provides other related advantages.